Composition and method for treating graft-versus-host disease

ABSTRACT

Compositions and methods are provided for prevention and clinical treatment of various forms of graft-versus-host disease (GVHD) by using inhibitors of adenosine deaminase (ADA). In particular, various formulations and dosing regimens of ADA inhibitors such as pentostatin are provided for the treatment of all forms of GVHD, especially steroid-refractory acute and chronic GVHD.

CROSS-REFERENCE

This application is a divisional application of Ser. No. 09/976,468,filed Oct. 12, 2001, which is incorporated herein by reference in itsentirety, and to which application we claim priority under 35 USC §121.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to compositions and methods for the treatment ofgraft-versus-host disease, and more specifically to the administrationof inhibitors of adenosine deaminase such as pentostatin and analogs andderivatives thereof.

2. Description of Related Art

For dysfunctional and/or diseased organs of the body, besidestherapeutic invention with drugs, organ transplantation is analternative, sometimes the last resort in the treatment of the patient.Particularly for patients with leukemia, end-stage renal, cardiac,pulmonary or hepatic failure, organ transplantation is quite commonlyused in the treatment. For example, allografts (organ grafts harvestedfrom donors other than the patient him/herself or host/recipient of thegraft) of various types, e.g. kidney, heart, lung, liver, bone marrow,pancreas, cornea, small intestine and skin (e.g. epidermal sheets) arecurrently routinely performed. Xenografts (organ grafts harvested fromnon-human animals), such as porcine heart valves, are also being usedclinically to replace their dysfunctional human counterparts.

To ensure successful organ transplantation, it is desirable to obtainthe graft from the patient's identical twin or his/her immediate familymember. This is because organ transplants evoke a variety of immuneresponses in the host, which results in rejection of the graft andgraft-versus-host disease (hereinafter, referred to as “GVHD”).

The immune response is primarily triggered by T cells throughrecognition of alloantigens, and the major targets in-transplantrejection are non-self allelic forms of class I and class II MajorHistocompatibility Complex (MHC) antigens. In acute rejection, donor'santigen-presenting cells such as dendritic cells and monocytes migratefrom the allograft to the regional lymph nodes, where they arerecognized as foreign by the recipient's CD4⁺ T_(H) cells, stimulatingT_(H) cell proliferation. Following T_(H) cells proliferation, apopulation of effector cells (including cytotoxic CD8⁺ T cells and CD4⁺T cells) is generated, which migrates and infiltrates to the graft andmediates graft rejection (Noelle et al. (1991) FASEB 5(13):2770).

Whereas acute rejection is a T cell-dependent process, a broad array ofeffector mechanisms participates in graft destruction. Through therelease of cytokines and cell-to-cell interactions, a diverse assemblyof lymphocytes including CD4⁺ T cells, CD8⁺ cytotoxic T cells,antibody-forming B cells and other proinflammatory leukocytes, isrecruited into the anti-allograft response. Antigen-presenting graftcells are destroyed directly by cytotoxic CD8⁺ T cells. Activated CD4⁺ Tcells produce interleukin-2 (hereinafter, referred to as “IL-2”), whichis essential to the activation of both CD8⁺ T cells and B cells.Additionally, CD4⁺ T cells produce other cytokines such as IFN-γ andIL-4 that also contribute to the destruction of allograft. Furthermore,interferon-γ (hereinafter, referred to as “IFN-γ”) induces increasedexpression of class I and class II MHC molecules on graft tissue, whichis more readily attacked by alloreactive effector cells. IFN-γ enhancesmacrophage activity and affects many inflammatory cells leading todelayed-type-hypersensitivity reaction and inflammation causingnonspecific damage to the graft. These reactions appear to be theprimary cause of the early acute rejection that may occur within thefirst few weeks after transplant. If untreated, acute rejectionprogresses to a rapid and severe process that causes destruction of thetransplant within a few days.

On the other hand, when a T-lymphocyte from the donor recognizes thedifferences based on a set of genetic markers, generally referred to ashuman leukocyte antigens (HLA), and it starts to attack the new body,i.e., the patient's body. Although most patients and donors are matchedas closely as possible for HLA markers. Many minor markers, however,differ between donors and patients except when the patient and donor areidentical twins. Before a transplant, extensive typing of the donor andrecipient is performed to make sure that the donor and recipient arevery close immunologically.

Despite this typing there are immunological differences that cannot bedetected and that the T-lymphocytes in the donor graft are capable ofdetecting. As a result, the donor T-lymphocytes start to attack thepatient's body and cause GVHD.

There are two forms of GVHD: the acute and chronic GVHD. Acute GVHDusually occurs within the first three months following a transplant.T-cells present in the donor's bone marrow at the time of transplantattack the patient's skin, liver, stomach, and/or intestines. Theearliest signs of acute GVHD are usually a skin rash that appears on thehand, feet and face. Other than blistering skin, patients with severeGVHD also develop large amounts of watery or bloody diarrhea withcramping due to the donor's T-cells' attack on the stomach andintestines. Jaundice (yellowing of the skin and eyes) is the usualindication that GVHD disease involves the liver. The more organsinvolved and the worse the symptoms, the worse the GVHD disease.

In the case of bone marrow transplantation, in particular, GVHD isanother obstacle to survival of transplanted patients. Storb (1984)“Pathophysiology and prevention of graft-versus-host disease.” InAdvances in Immunobiology: Blood cell antigens and bone marrowtransplantation, McCullogh and Sandier, editors, Alan, Inc., N.Y., p.337. A large proportion of GVHD-afflicted individuals dies as a resultof GVHD. Weiden et al. (1980) “Graft-versus-host disease in allogeneicmarrow transplantation”, in Biology of Bone-Marrow Transplantation, Galeand Fox, editors, Academic Press, N.Y., p 37.

To protect patients from such fatal damages, various immunosuppressiveagents have been employed. Currently, allograft rejection is controlledusing immunosuppressive agents such as cyclosporin A, azathioprine,corticosteroids including prednisone, and methylprednisolone,cyclophosphamide, and FK506. Cyclosporin A, the most powerful and mostfrequently used immunosuppressant, revolutionized the field of organtransplant surgery. Other immunosuppressive agents such as FK506,rapamycin, mycophenolic acid, 15-deoxyspergualin, mimoribine,misoprostol, OKT3 and anti-IL-2 receptor antibodies, have been used inthe treatment and/or prevention of organ transplantation rejection.Briggs, Immunology letters, 29(1-2), 89-94, 1991; FASEB 3:3411, 1989.Although the development of new immunosuppressive drugs has led tosubstantial improvement in the survival of patients, these drugs areassociated with a high incidence of side effects such as nephrotoxicityand/or hepatotoxicity.

For example, cyclosporin A has associated toxicities and side effectswhen used even at therapeutic doses. Although FK506 is about 10 to 100times more potent than cyclosporin A in inhibiting activation-inducedIL-2 transcription in vitro and graft rejection in vivo, it also showsside effects such as neurotoxicity and nephrotoxicity. Thus, there stillexists the need for treatment and prophylaxis for GVHD with improvedtoxicity profiles.

SUMMARY OF THE INVENTION

Compositions and methods are provided for prevention and clinicaltreatment of various forms of graft-versus-host disease (GVHD) by usinginhibitors of adenosine deaminase (ADA). In particular, novelformulations and dosing regimens of ADA inhibitors such as pentostatinare provided for the treatment of humans in vivo as well as for ex vivoconditioning of organ transplants in order to specifically suppressT-lymphocyte mediated immune responses while minimizing systemictoxicity of the drug.

In one aspect, a method is provided for treating a patient havinggraft-versus-host disease. The method comprises: administering to thepatient an adenosine deaminase (ADA) inhibitor in a pharmaceuticallyeffective amount. Examples of the adenosine deaminase inhibitor include,but are not limited to, pentostatin, fludarabine monophosphate, andcladribine. The ADA inhibitor may be administered orally or parenterally(e.g., via intravenous infusion or injection) to the patient.

The patient may have acute or chronic graft-versus-host disease, and mayhave also failed at least one immunosuppressive regimen such as aregimen including steroids (e.g., prednisone and methylprednisolone),cyclophosphamide, cyclosporin A, FK506, thalidomide, azathioprine, anddaclizumab.

In one embodiment, the method is used to treat hematopoietic stem celltransplant (HSCT) patients manifesting grade 2 or greater acute GVHD,who have failed to respond to treatment with at least 2 mg/Kg ofmethylprednisolone or equivalent corticosteroid or other salvagetherapy. For example, the HSCT patient may be treated with pentostatinat 0.25-1 mg/m²/day as a 20 minute intravenous (IV) infusion on days 1,2 and 3.

The method may further comprise: monitoring the improvement of the GVHDsymptoms in the skin, mouth, fascia, and liver. Treatment withpentostatin may be repeated to further reduce the symptoms or to preventrecurrence of the disease.

In another embodiment, the method is used to treat steroid-refractorychronic graft vs host disease (cGVHD). For example, recipients ofallogeneic HSCT developing cGVHD who have failed to respond to treatmentwith at least 2 mg/Kg of methylprednisolone or equivalent corticosteroidor other salvage therapy may be treated with pentostatin. For example,pentostatin may be orally administrated to the chronic GVHD patient at adose between about 1-10 mg/m², preferably between about 2-6 mg/², andmore preferably between about 2-4 mg/m² each day for 3 consecutive dayseach month.

In another aspect, a method is provided for preventing or reducing therisk of developing graft-versus-host disease in a recipient of an organor tissue transplant. The method comprises: administering to thetransplant recipient an adenosine deaminase (ADA) inhibitor in apharmaceutically effective amount within a predetermined time windowbefore or after the transplantation. Examples of the ADA inhibitorinclude, but are not limited to, pentostatin, fludarabine monophosphate,and cladribine. The ADA inhibitor may be administered orally orparenterally (e.g., via intravenous infusion or injection) to therecipient of organ transplantation.

In one embodiment, pentostatin is administered orally to the transplantrecipient 3 or 2 days before the transplantation. Alternatively,pentostatin may be administered to the transplant recipient by IVinfusion at a dose lower than 2 mg/m², preferably at a dose lower than 1mg/m².

In a variation of the embodiment, the transplant recipient istransplanted with hematopoietic stem cells and treated in amyeloablative conditioning regimen. The recipient may be treated withpentostatin via oral administration at about 0.5-2.0 mg/m² on days −14,−13, −12 and −3, −2, −1 with high dose cyclophosphamide and/or busulfanand/or melphalan and/or 1200-1800 cGy irradiation prior to stem cellinfusion. Post transplantation the recipient may be continuously treatedwith pentostatin, on days +8 and +15 preferably iv at a lower dose suchas 1-2 mg/m².

In another embodiment, pentostatin may be administered to a transplantrecipient after the transplantation. For example, for standard (i.e.,myeloablative) transplant-or non-myeloablative stem cell transplant(NST) where pentostatin is not used in the conditioning regimen,pentostatin is administered to the transplant recipient at 0.5-1.5mg/m²/day on days +8, +15, +22 and +30 following stem cell infusion.

The above methods may further comprise: administering to the GVHDpatient or the transplant recipient an immunosuppressive agent selectedfrom the group consisting of prednisone, methylprednisolone,cyclophosphamide, cyclosporin A, FK506, thalidomide, azathioprine,Daclizumab, Infliximab, MEDI-205, abx-cbl and ATG.

In yet another aspect, a method is provided for ex vivo or in vitrotreatment of blood derived cells, bone marrow transplants, or otherorgan transplants. The method comprises: treating a tissue or organtransplant with an ADA inhibitor in an effective amount such thatactivity of T-lymphocytes therein is substantially inhibited, preferablyby at least 50% reduction in activity, more preferably by at least 80%reduction in activity, and most preferably by at least 90% reduction inactivity.

Examples of the tissue or organ transplant include, but are not limitedto, stem cells, bone marrow, heart, liver, kidney, lung, pancreas, smallintestine, cornea, and skin. Examples of the ADA inhibitor include, butare not limited to, pentostatin, fludarabine monophosphate, andcladribine.

In one embodiment, the transplant is stored in a preservation solutioncontaining pentostatin in an amount sufficient to inhibit activity ofT-lymphocytes of the transplant. An example of commercially availablepreservation solutions is Plegisol (Abbott). The preservation solutionmay also contain conventional co-solvents, excipients, stabilizingagents and/or buffering agents.

In another embodiment, the transplant is washed with a buffer containingpentostatin prior to storage or transplantation. In this way, the riskof developing acute GVHD upon transplantation should be significantlyreduced, and the host is not only protected from GVHD but also frompotential side effects of pentostatin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for prevention and clinicaltreatment of various forms of graft-versus-host disease (GVHD) by usinginhibitors of adenosine deaminase (ADA). In particular, novelformulations and dosing regimens of ADA inhibitors such as pentostatinare provided for the treatment in order to specifically suppressT-lymphocyte mediated immune responses while minimizing systemictoxicity of the drug, especially myelosuppression.

In essence, the methods operate by exploiting the effects of certainadenosine analogs such as pentostatin on ADA-dependent T-lymphocytes. Itis believed that pentostatin asserts potent inhibitory effects on ADA,which ultimately leads to apoptosis of T-lymphocytes (i.e., T-cells).However, physiological functions of B-cells that are T-cell independentand hemopoietic stem cells are largely unaffected. By exploiting thepreferential toxic effects of an ADA inhibitor on T-cells, patients canbe treated with the drug prior to or within a time window posttransplantation to prevent onset of GVHD or organ rejection. Further,patients with acute or chronic GVHD can be treated with the drug toreduce the pathological symptoms with minimal myelosuppression. Inaddition, the ADA inhibitor may be used for ex vivo treatment orconditioning of transplants to specifically activate T-lymphocytes fromthe donor without substantially affecting rate of engraftment.

The following sections describe in detail the mechanisms of actions ofthe ADA-inhibitors, their formulations and methods of the use forprevention and clinical treatment of GVHD, and for ex vivo conditioningof the transplants

1. Adenosine Deaminase (ADA) and its Inhibitors

ADA is a 41 kD protein expressed in all tissues with highest expressionin lymphocytes. ADA participates in the purine metabolism where itdegrades either adenosine or 2′-deoxyadenosine producing inosine or2′-deoxyinosine, respectively. It has been found that the activity ofADA is subject to changes depending upon the degree of activity of thecell, i.e. whether differentiation or proliferation occurs (Trotta, P.P. and Balis M. E. (1977) Cancer Research 37: 2297-2305). A geneticdeficiency of ADA may cause severe combined immunodeficiency. Dighiero,G. (1996) “Adverse and beneficial immunological effects of purinenucleoside analogues,” Hematol Cell Ther, 38:575-581.

Certain adenosine analogs have been found to have inhibitory effects onADA. These compounds include, but are not limited to, pentostatin(2′-deoxycoformycin, also referred to as dCF, or NIPENT®); fludarabinemonophosphate (FLU), a fluorinated analogue of adenine that isrelatively resistant to adenosine-deaminase, and cladribine(2-chloro-2′-deoxyadenosine or 2CDA) which is also resistant toadenosine deaminase through introduction of a chlorine at the 2 carbon.These adenosine analogs that inhibits ADA activity and/or cause abnormalaccumulation of deoxyadenosine and adenosine in the cell are hereincollectively referred to as “ADA inhibitors”.

While the exact nature of the ADA pathway intervention by these ADAinhibitors seems unclear, it may be that these compounds that areresistant to cellular deamination might mimic the ADA-deficient state.Lack of ADA seems to lead to a build up of deoxyadenosine and adenosinetriphosphate in the cell, thus fatally accelerating DNA strand breaks inthe cell. Under normal conditions, cells are continuously breaking andrejoining DNA. When this physiological process is accelerated by theeffect of excess adenosine triphosphate, it leads to consumption of NADfor poly-ADP-ribose synthesis. This polymer is produced fromnicotinamide adenosine dinucleotides (NAD) in a reaction catalyzed bythe chromatin-associated poly(ADP-ribose) synthetase, leading to adepletion of the NAD content of the cell. This depletion induces aprofound alteration of cellular reducing power, because of lethal ADPand ATP depletion.

The result is programmed cell death through activation of a Ca²⁺−,Mg²⁺-dependent endonuclease. Hence, it appears that the ADA inhibitorsaccording to the invention can act on cells, with preferentiallymphocytic activity, via an apoptotic process. The fact thatsupplementation of a cell medium with the NAD precursor of nicotinamideor 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthetase,prevented NAD depletion and reduces 2CDA toxicity, tends to support thishypothesis.

The ADA inhibitors listed above may affect the ADA pathway in differentmanners but ultimately leads accumulation of adenosine triphosphate andpromotes apoptosis of cells, especially lymphocytes that have low levelsof the nucleoside-cleaving enzyme 5′-nucleotidase are particularlysensitive to these antimetabolic effects. Pentostatin, for example, hasbeen shown to be an irreversible inhibitor of ADA. By favoring thepredominance of deoxycytidine kinase (DCK) over the dephosphorylatingenzyme 5-nucleotidase in lymphocytes it induces a preferentialaccumulation of deoxyadenosine-5′-triphosphate (dATP). By comparison,FLU and 2CDA are rather resistant to the enzyme. Both drugs areinitially phosphorylated by DCK and contribute to the accumulation ofcellular adenosine triphosphate surrogates. As noted above, theaccumulation of adenosine triphosphate, whether by the presumedpentostatin mechanism, or the FLU or 2CDA mechanism, promotes theapoptotic death of the cell.

Pentostatin has been widely used as an antimetabolite to treat variousforms of leukemia in the clinic. C. Dearden, et al., “Deoxycoformycin inthe treatment of mature T-cell leukemias”, Brit J. of Can.,64(5):903-906 (November 1991); J. Seymour et al., “Response duration andrecovery of CD4+ lymphocytes following deoxycoformycin ininterferon-α-resistant hairy cell leukemia: 7-year follow-up”, Leukemia,11, 42-47 (1997); J. Johnston et al., “Induction of Apoptosis in CD4+Prolymphocytic Leukemia by Deoxyadenosine and 2′-Deoxycoformycin”,Leukemia Research, 16:8, 781-788 (1992); E. Copelan et al.,“Pharmacologic Marrow Purging in Murine T Cell Leukemia”, Blood,71(6):1656-1661 (June 1988).

According to the present invention, it is believed that ADA inhibitorscan be used to preferentially targeting T-lymphocytes, including thosefrom the donor. By reducing T-lymphocytes presumably through apoptosiscaused by the ADA inhibitor, attack of the donor T-lymphocytes on thehost's organs may be prevented or significantly compromised.

2. Formulations of ADA Inhibitors

The present invention provides novel formulations and dosing regimens ofADA inhibitors for treating GVHD. By using the methodology of thepresent invention, GVHD may be treated in a more efficacious andconvenient way and with a more favorable myelotoxicity profile. In apreferred embodiment, the ADA inhibitor is delivered orally for thetreatment or prevention of GVHD. Optionally, the ADA inhibitor may bedelivered mucosally or nasally for the treatment or prevention of GVHD.Alternatively, the ADA inhibitor may be delivered parenterally, inparticular, intravenously. Preferably, the ADA inhibitor is delivered ata lower dose than those used in an oncological treatment.

1) ADA Inhibitors

The ADA inhibitor referred within is a composition that has aninhibitory effect on biochemical activity of adenosine deaminase (ADA).Examples of the ADA inhibitor include, but are not limited to,pentostatin, fludarabine monophosphate, and cladribine. Other ADAinhibitor may be adenosine analogs that compete with adenosine forbinding to ADA such as 2-deoxyadenosine, 3′-deoxyadenosine, anddideoxyadenosine.

Optionally, the ADA inhibitor is in the form of a therapeuticallyacceptable salt. This salt may be prepared in the conventional manner.Salt formers that may, for example, be used are conventional anions orsalts thereof that are physiologically acceptable in the salt form.Examples thereof are: amino acids such as tyrosine or asparagine,sulfates, phosphates, carboxylic acids, tosylates, nitrates, acetates,and long chain fatty acid derivatives of these.

Should the ADA inhibitor be used in the form of a salt, the salt formermay also be used in excess, i.e. in an amount greater than equimolar.

2) Oral Formulation of ADA Inhibitors

It one aspect, the ADA inhibitor is formulated for oral administration.Currently, pentostatin, for example, that is used in the clinic fortreating hairy cell leukemia is formulated for intravenous (IV)administration. There are a few practical limitations associated withsuch a dosage form. For example, IV dosing is expensive. It requires ahighly trained medical professional to administer the IV dose. Thedosing involves expensive equipment and materials. Additionally, IVdosing presents increased possibilities of infection, through use ofcontaminated equipment or accidental-contamination, for example. This isa special concern in health care settings where increased incidences ofantibiotic resistant bacteria are being noted.

An oral dosage form may alleviate most, if not all, of theabove-mentioned problems associated with IV or other parenteral dosageforms. However, it is well recognized in the art that deoxyadenosineanalogs such as pentostatin is highly acid-labile. They are highlysusceptible to acid-catalyzed glycosidic cleavage. Therefore, one ofordinary skill in the art would expect that an orally administeredadenosine analog would be cleaved in the stomach, and rendered inactive.In fact, investigators studying pentostatin, have not considered oraladministration of the drug worth studying because of its known acidlability. Marvin M. Chassin et al. “Enzyme Inhibition Titration Assayfor 2′-deoxycoformycin and its Application to the Study of theRelationship Between Drug Concentration and Tissue Adenosine Deaminasein Dogs and Rats” Biochemical Pharmacology 28:1849-1855 (1979).Likewise, other researchers have reported on the acid lability of2′-deoxycoformycin. L. A. al-Razzak et al. “Chemical Stability ofPentostatin (NSC-218321), a Cytotoxic and Immunosuppressant Agent”,Pharm. Res. 7:452-460 (1990).

Other ADA inhibitors may be expected to have similar acid labilitycharacteristics. A. Tarasiuk et al. “Stability of2-chloro-2′-deoxyadenosine at Various pH and Temperature” Arch. Immunol.Ther. Exp. (Warsz) 42:13-15 (1994); T. Ono “2′-Fluoro Modified NucleicAcids: Polymerase-directed Synthesis, Properties and Stability toAnalysis by Matrix-Assisted Laser Desorption/Ionization MassSpectrometry” Nucleic Acids Res. 25:4581-4588 (1997).

Against this mainstream of thought the inventors believe that the ADAinhibitors can be formulated for oral administration and convenientlyused to treat various forms of GVHD. It is also believed that it ispossible to achieve bioavailability of the ADA inhibitor using an oraldosage form and the effects of the drug on the patient in an oral dosageform should be reasonably close to those achieved using an IV dosageform.

Various oral dosage forms of the ADA inhibitor may be used in thepractice of the invention. For example, the ADA inhibitor may be mixedwith a pharmacologically acceptable liquid and swallowed. The ADAinhibitor also may be compounded into tablets, capsules, pills,lozenges, etc. using conventional compounding techniques.

In one embodiment, the ADA inhibitor may be administered with variousagents to reduce acid concentration in the stomach. This reduces acidlability and allows for enhanced concentrations of the ADA inhibitor forenhanced gastric and/or intestinal absorption. For example, theadenosine analog may be coadministered with an H2 inhibitor such ascimetidine, an acid neutralizer such as calcium carbonate, or a protonpump inhibitor.

Furthermore, the ADA inhibitor may be (co)administered using a dosageform that reduces the effect of acid lability on their bioavailability.(Co)administration within the context of this invention may be taken tomean administration, coadministration, or both. Coadministration in thecontext of this invention may be defined to mean the administration ofmore than one therapeutic in the course of a coordinated treatment toachieve an improved clinical outcome. Such coadministration may also becoextensive, that is, occurring during overlapping periods of time.

In one variation, the ADA inhibitor such as pentostatin is formulated ina dosage form that reduces acid lability of the compound, therebyenhancing the bioavailability the compound. For example, pentostatin maybe compounded with polymer matrices that are erodible chemically orbiologically.

Optionally, the ADA inhibitor may be coated with an enteric coating toprevent ready decomposition in the stomach. The enteric coating maycomprise hydroxypropyl-methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymer, polyvinyl acetate-phthalate andcellulose acetate phthalate.

The dosage form of the ADA inhibitor may also be a solid dispersion witha water soluble or a water insoluble carrier. Examples of water solubleor water insoluble carrier include, but are not limited to, polyethyleneglycol, polyvinylpyrrolidone, hydroxypropylmethyl-cellulose,phosphatidylcholine, polyoxyethylene hydrogenated castor oil,hydroxypropylmethylcellulose phthalate, carboxymethylethylcellulose, orhydroxypropylmethylcellulose, ethyl cellulose, or stearic acid.

Optionally, oral dosage form of the ADA inhibitor may be a complexbetween an ion exchange resin and the ADA inhibitor.

In another aspect, the ADA inhibitor may be formulated for a controlledrelease. Controlled release within the scope of this invention can betaken to mean any one of a number of extended release dosage forms.

The following terms may be considered to be substantially equivalent tocontrolled release, for the purposes of the present invention:continuous release, controlled release, delayed release, depot, gradualrelease, long-term release, programmed release, prolonged release,proportionate release, protracted release, repository, retard, slowrelease, spaced release, sustained release, time coat, timed release,delayed action, extended action, layered-time action, long acting,prolonged action, repeated action, slowing acting, sustained action,sustained-action medications, and extended release. Further discussionsof these terms may be found in Lesczek Krowczynski “Extended-ReleaseDosage Forms”, 1987 (CRC Press, Inc.).

The various controlled release technologies cover a very broad spectrumof drug dosage forms. Controlled release technologies include, but arenot limited to physical systems and chemical systems. Physical systemsinclude, but not limited to, reservoir systems with rate-controllingmembranes, such as microencapsulation, macroencapsulation, and membranesystems; reservoir systems without rate-controlling membranes, such ashollow fibers, ultra microporous cellulose triacetate, and porouspolymeric substrates and foams; monolithic systems, including thosesystems physically dissolved in non-porous, polymeric, or elastomericmatrices (e.g., non-erodible, erodible, environmental agent ingression,and degradable), and materials physically dispersed in non-porous,polymeric, or elastomeric matrices (e.g., non-erodible, erodible,environmental agent ingression, and degradable); laminated structures,including reservoir layers chemically similar or dissimilar to outercontrol layers; and other physical methods, such as osmotic pumps, oradsorption onto ion-exchange resins.

Chemical systems include, but are not limited to, chemical erosion ofpolymer matrices (e.g., heterogeneous, or homogeneous erosion), orbiological erosion of a polymer matrix (e.g., heterogeneous, orhomogeneous). Additional discussion of categories of systems forcontrolled release may be found in Agis F. Kydonieus, “ControlledRelease Technologies: Methods, Theory and Applications” 1980 (CRC Press,Inc.).

Controlled release drug delivery systems may also be categorized undertheir basic technology areas, including, but not limited to,rate-preprogrammed drug delivery systems, activation-modulated drugdelivery systems, feedback-regulated drug delivery systems, andsite-targeting drug delivery systems.

In rate-preprogrammed drug delivery systems, release of drug moleculesfrom the delivery systems “preprogrammed” at specific rate profiles.This may be accomplished by system design, which controls the moleculardiffusion of drug molecules in and/or across the barrier medium withinor surrounding the delivery system. Fick's laws of diffusion are oftenfollowed.

In activation-modulated drug delivery systems, release of drug moleculesfrom the delivery systems is activated by some physical, chemical orbiochemical processes and/or facilitated by the energy suppliedexternally. The rate of drug release is then controlled by regulatingthe process applied, or energy input.

In feedback-regulated drug delivery systems, release of drug moleculesfrom the delivery systems may be activated by a triggering event, suchas a biochemical substance, in the body. The rate of drug release isthen controlled by the concentration of triggering agent detected by asensor in the feedback regulated mechanism.

In a site-targeting controlled-release drug delivery system, the drugdelivery system targets the active molecule to a specific site or targettissue or cell. This may be accomplished, for example, by a conjugateincluding a site specific targeting moiety that leads the drug deliverysystem to the vicinity of a target tissue (or cell), a solubilizer thatenables the drug delivery system to be transported to and preferentiallytaken up by a target tissue, and a drug moiety that is covalently bondedto the polymer backbone through a spacer and contains a cleavable groupthat can be cleaved only by a specific enzyme at the target tissue.

While a preferable mode of controlled release drug delivery will beoral, other modes of delivery of controlled release compositionsaccording to this invention may be used to treat or prevent GVHD. Theseinclude mucosal delivery, nasal delivery, ocular delivery, transdermaldelivery, parenteral controlled release delivery, vaginal delivery,rectal delivery and intrauterine delivery. All of these dosage forms maybe manufactured using conventional techniques, together with thetechniques discussed herein.

There are a number of controlled release drug formulations that aredeveloped preferably for oral administration. These include, but are notlimited to, osmotic pressure-controlled gastrointestinal deliverysystems; hydrodynamic pressure-controlled gastrointestinal deliverysystems; membrane permeation-controlled gastrointestinal deliverysystems, which include microporous membrane permeation-controlledgastrointestinal delivery devices; gastric fluid-resistant intestinetargeted controlled-release gastrointestinal delivery devices; geldiffusion-controlled gastrointestinal delivery systems; andion-exchange-controlled gastrointestinal delivery systems, which includecationic and anionic drugs. Additional information regarding controlledrelease drug delivery systems may be found in Yie W. Chien “Novel DrugDelivery Systems” 1992 (Marcel Dekker, Inc.). Some of these formulationswill now be discussed in more detail.

Enteric coatings may be applied to tablets to prevent the release ofdrugs in the stomach either to reduce the risk of unpleasant sideeffects or to maintain the stability of the drug which might otherwisebe subject to degradation due to expose to the gastric environment. Mostpolymers that are used for this purpose are polyacids that function byvirtue of the fact that their solubility in aqueous medium ispH-dependent, and they require conditions with a pH higher than normallyencountered in the stomach.

Enteric coatings may be used to coat a solid or liquid dosage form ofadenosine analogs according to the invention. Enteric coatings promotethe inventive adenosine analogs remaining physically incorporated in thedosage form for a specified period when exposed to gastric juice. Yetthe enteric coatings are designed to disintegrate in intestinal fluidfor ready absorption. Delay of the adenosine analogs' absorption isdependent on the rate of transfer through the gastrointestinal tract,and so the rate of gastric emptying is an important factor. Someinvestigators have reported that a multiple-unit type dosage form, suchas granules, may be superior to a single-unit type. Therefore, in apreferable embodiment, the ADA inhibitor according to the invention maybe contained in an enterically coated multiple-unit dosage form. In amore preferable embodiment, the dosage form of the ADA inhibitoraccording to the invention is prepared by spray-coating granules of anadenosine analog-enteric coating agent solid dispersion on an inert corematerial:. These granules can result in prolonged absorption of the drugwith good bioavailability.

Typical enteric coating agents include, but are not limited to,hydroxypropylmethylcellulose phthalate, methacrylic acid-methacrylicacid ester copolymer, polyvinyl acetate-phthalate and cellulose acetatephthalate. Akihiko Hasegawa “Application of solid dispersions ofNifedipine with enteric coating agent to prepare a sustained-releasedosage form” Chem. Pharm. Bull. 33: 1615-1619 (1985). Various entericcoating materials may be selected on the basis of testing to achieve anenteric coated dosage form designed ab initio to have a preferablecombination of dissolution time, coating thicknesses and diametralcrushing strength. S. C. Porter et al. “The Properties of Enteric TabletCoatings Made From Polyvinyl Acetate-phthalate and Cellulose acetatePhthalate”, J. Pharm. Pharmacol. 22:42p (1970).

On occasion, the performance of an enteric coating may hinge on itspermeability. S. C. Porter et al., .“The Permeability of EntericCoatings and the Dissolution Rates of Coated Tablets”, J. Pharm.Pharmacol. 34: 5-8 (1981). With such oral drug delivery systems, thedrug release process may be initiated by diffusion of aqueous fluidsacross the enteric coating. Investigations have suggested osmoticdriven/rupturing affects as important release mechanisms from entericcoated dosage forms. Roland Bodmeier et al. “Mechanical Properties ofDry and Wet Cellulosic and Acrylic Films Prepared from Aqueous ColloidalPolymer Dispersions used in the Coating of Solid Dosage Forms”Pharmaceutical Research, 11: 882-888 (1994).

Another type of useful oral controlled release structure is a soliddispersion. A solid dispersion may be defined as a dispersion of one ormore active ingredients in an inert carrier or matrix in the solid stateprepared by the melting (fusion), solvent, or melting-solvent method.Akihiko Hasegawa “Super Saturation Mechanism of Drugs from SolidDispersions with Enteric Coating Agents”, Chem. Pharm. Bull. 36:4941-4950 (1998). The solid dispersions may be also called solid-statedispersions. The term “coprecipitates” may also be used to refer tothose preparations obtained by the solvent methods.

Solid dispersions may be used to improve the solubilities and/ordissolution rates of the ADA inhibitor according to the invention thatmay be poorly water-soluble. Hiroshi Yuasa, et al, “Application of theSolid Dispersion Method to the Controlled Release Medicine. III. Controlof the Release Rate of Slightly Water-Soluble Medicine From SolidDispersion Granules” Chem. Pharm. Bull. 41:397-399 (1993). The soliddispersion method was originally used to enhance the dissolution rate ofslightly water-soluble medicines by dispersing the medicines intowater-soluble carriers such as polyethylene glycol orpolyvinylpyrrolidone, Hiroshi Yuasa, et al. “Application of the SolidDispersion Method to the Controlled Release of Medicine. TV. PreciseControl of the Release Rate of a Water-Soluble Medicine by Using theSolid Dispersion Method Applying the Difference in the Molecular Weightof a Polymer” Chem. Pharm. Bull. 41:933-936 (1993).

The selection of the carrier may have an influence on the dissolutioncharacteristics of the dispersed drug because the dissolution rate of acomponent from a surface may be affected by other components in amultiple component mixture. For example, a water-soluble carrier mayresult in a fast release of the drug from the matrix, or a poorlysoluble or insoluble carrier may lead to a slower release of the drugfrom the matrix. The solubility of poorly water soluble adenosineanalogs according to the invention may also be increased owing to someinteraction with the carriers.

Examples of carriers useful in solid dispersions according to theinvention include, but are not limited to, water-soluble polymers suchas polyethylene glycol, polyvinylpyrrolidone, orhydroxypropylmethyl-cellulose. Akihiko Hasegawa, “Application of SolidDispersions of Nifedipine with Enteric Coating Agent to Prepare aSustained-release Dosage Form” Chem. Pharm. Bull. 33: 1615-1619 (1985).

Alternate carriers include phosphatidylcholine. Makiko Fuji, et al. “TheProperties of Solid Dispersions of Indomethacin, Ketoprofen andFlurbiprofen in Phosphatidylcholine” Chem. Pharm. Bull; 36:2186-2192(1988). Phosphatidylcholine is an amphoteric but water-insoluble lipid,which may improve the solubility of otherwise insoluble adenosineanalogs in an amorphous state in phosphatidylcholine solid dispersions.See Makiko Fuji, et al., “Dissolution of Bioavailability of Phenytoin inSolid Dispersion with Phosphatidylcholine” Chem. Pharm. Bull36:4908-4913 (1988).

Other carriers include polyoxyethylene hydrogenated castor oil.Katsuhiko Yano, et al. “In-Vitro Stability and In-Vivo AbsorptionStudies of Colloidal Particles Formed From a Solid Dispersion System”Chem. Pharm. Bull 44:2309-2313 (1996). Poorly water-soluble adenosineanalogs according to the invention may be included in a solid dispersionsystem with an enteric polymer such as hydroxypropylmethylcellulosephthalate and carboxymethylethylcellulose, and a non-enteric polymer,hydroxypropylmethylcellulose. See Toshiya Kai, et al., “Oral AbsorptionImprovement of Poorly Soluble Drug Using Soluble Dispersion Technique”,Chem. Pharm. Bull. 44:568-571 (1996). Another solid dispersion dosageform includes incorporation of the ADA inhibitor with ethyl celluloseand stearic acid in different ratios. Kousuke Nakano, et al. “OralSustained-Release Cisplatin Preparations for Rats and Mice” J. Pharm.Pharmacol. 49:485-490 (1997).

There are various methods commonly known for preparing soliddispersions. These include, but are not limited to the melting method,the solvent method and the melting-solvent method.

In the melting method, the physical mixture of a drug in a water-solublecarrier is heated directly until it melts. The melted mixture is thencooled and solidified rapidly while rigorously stirred. The final solidmass is crushed, pulverized and sieved. Using this method a supersaturation of a solute or drug in a system can often be obtained byquenching the melt rapidly from a high temperature. Under suchconditions, the solute molecule may be arrested in solvent matrix by theinstantaneous solidification process. A disadvantage is that manysubstances, either drugs or carriers, may decompose or evaporate duringthe fusion process at high temperatures. However, this evaporationproblem may be avoided if the physical mixture is heated in a sealedcontainer. Melting under a vacuum or blanket of an inert gas such asnitrogen may be employed to prevent oxidation of the drug or carrier.

The solvent method has been used in the preparation of solid solutionsor mixed crystals of organic or inorganic compounds. Solvent methoddispersions may be prepared by dissolving a physical mixture of twosolid components in a common solvent, followed by evaporation of thesolvent. The main advantage of the solvent method is that thermaldecomposition of drugs or carriers may be prevented because of the lowtemperature required for the evaporation of organic solvents. However,some disadvantages associated with this method are the higher cost ofpreparation, the difficulty in completely removing liquid solvent, thepossible adverse effect of its supposedly negligible amount of thesolvent on the chemical stability of the drug.

Another method of producing solid dispersions is the melting-solventmethod. It is possible to prepare solid dispersions by first dissolvinga drug in a suitable liquid solvent and then incorporating the solutiondirectly into a melt of polyethylene glycol, obtainable below 70degrees, without removing the liquid solvent. The selected solvent ordissolved adenosine analogs may be selected such that the solution isnot miscible with the melt of polyethylene glycol. The polymorphic formof the adenosine analogs may then be precipitated in the melt. Such aunique method possesses the advantages of both the melting and solventmethods. Win Loung Chiou, et al. “Pharmaceutical Applications of SolidDispersion Systems” J. Pharm. Sci. 60:1281-1301 (1971).

Another-controlled release dosage form is a complex between an ionexchange resin and the ADA inhibitor according to the invention. Ionexchange resin-drug complexes have been used to formulatesustained-release products of acidic and basic drugs. In one-preferableembodiment, a polymeric film coating is provided to the ion exchangeresin-drug complex particles, making drug release from these particlesdiffusion controlled. See Y. Raghunathan et al. “Sustained-released drugdelivery system I: Coded ion-exchange resin systems forphenylpropanolamine and other drugs” J. Pharm. Sciences 70: 379-384(1981).

Injectable micro spheres are another controlled release dosage form.Injectable micro spheres may be prepared by non-aqueous phase separationtechniques, and spray-drying techniques. Micro spheres may be preparedusing polylactic acid or copoly(lactic/glycolic acid). Shigeyuki Takada“Utilization of an Amorphous Form of a Water-Soluble GPIIb/IIIaAntagonist for Controlled Release From Biodegradable Micro spheres”Pharm. Res. 14:1146-1150 (1997), and ethyl cellulose, Yoshiyuki Koida“Studies on Dissolution Mechanism of Drugs from Ethyl CelluloseMicrocapsules” Chem. Pharm. Bull. 35:1538-1545 (1987).

Other controlled release technologies that may be used in the practiceof this invention are quite varied. They include SODAS, INDAS, IPDAS,MODAS, EFVAS, PRODAS, and DUREDAS. SODAS are multi particulate dosageforms utilizing controlled release beads. INDAS are a family of drugdelivery technologies designed to increase the solubility of poorlysoluble drugs. IPDAS are multi particulate tablet formation utilizing acombination of high density controlled release beads and an immediaterelease granulate. MODAS are controlled release single unit dosageforms. Each tablet consists of an inner core surrounded by asemipermeable multiparous membrane that controls the rate of drugrelease. EFVAS is an effervescent drug absorption system. PRODAS is afamily of multi particulate formulations utilizing combinations ofimmediate release and controlled release mini-tablets. DUREDAS is abilayer tablet formulation providing dual release rates within the onedosage form. Although these dosage forms are known to one of skill,certain of these dosage forms will now be discussed in more detail.

INDAS was developed specifically to improve the solubility andabsorption characteristics of poorly water soluble drugs. Solubilityand, in particular, dissolution within the fluids of thegastrointestinal tract is a key factor in determining the overall oralbioavailability of poorly water soluble drug. By enhancing solubility,one can increase the overall bioavailability of a drug with resultingreductions in dosage. INDAS takes the form of a high energy matrixtablet. In a preferred embodiment of the invention production involvesincluding adenosine analogs in an amorphous form together with acombination of energy, excipients, and unique processing procedures.

Once included in the desirable physical form, the resultant high energycomplex may be stabilized by an absorption process that utilizes a novelpolymer cross-linked technology to prevent recrystallization. Thecombination of the change in the physical state of the ADA inhibitoraccording to the invention coupled with the solubilizing characteristicsof the excipients employed enhances the solubility of the ADA inhibitoraccording to the invention. The resulting absorbed amorphous drugcomplex granulate may be formulated with a gel-forming erodable tabletsystem to promote substantially smooth and continuous absorption.

IPDAS is a multi-particulate tablet technology that may enhance thegastrointestinal tolerability of potential irritant and ulcerogenicdrugs. Intestinal protection is facilitated by the multi-particulatenature of the IPDAS formulation which promotes dispersion of an irritantADA inhibitor according to the invention throughout the gastrointestinaltract. Controlled release characteristics of the individual beads mayavoid high concentration of drug being both released locally andabsorbed systemically. The combination of both approaches serves tominimize the potential harm of the ADA inhibitor according to theinvention with resultant benefits to patients.

IPDAS is composed of numerous high density controlled release beads.Each bead may be manufactured by a two step process that involves theinitial production of a micromatrix with embedded adenosine analogsaccording to the invention and the subsequent coating of thismicromatrix with polymer solutions that form a rate limitingsemipermeable membrane in vivo. Once an IPDAS tablet is ingested, it maydisintegrate and liberate the beads in the stomach. These beads maysubsequently pass into the duodenum and along the gastrointestinaltract, preferably in a controlled and gradual manner, independent of thefeeding state. The release of the ADA inhibitor occurs by diffusionprocess through the micromatrix and subsequently through the pores inthe rate controlling semipermeable membrane. The release rate from theIPDAS tablet may be customized to deliver a drug-specific absorptionprofile associated with optimized clinical benefit. Should a fast onsetof activity be necessary, immediate release granulate may be included inthe tablet. The tablet may be broken prior to administration, withoutsubstantially compromising drug release, if a reduced dose is requiredfor individual titration.

MODAS is a drug delivery system that may be used to control theabsorption of a water soluble ADA inhibitor according to the invention.Physically MODAS is a non-disintegrating table formulation thatmanipulates drug release by a process of rate limiting diffusion by asemipermeable membrane formed in vivo. The diffusion process essentiallydictates the rate of presentation of drug to the gastrointestinalfluids, such that the uptake into the body is controlled. Because of theminimal use of excipients, MODAS can readily accommodate small dosagesize forms. Each MODAS tablet begins as a core containing active drugplus excipients. This core is coated with a solution of insolublepolymers and soluble excipients. Once the tablet is ingested, the fluidof the gastrointestinal tract may dissolve the soluble excipients in theouter coating leaving substantially the insoluble polymer. What resultsis a network of tiny, narrow channels connecting fluid from thegastrointestinal tract to the inner drug core of water soluble drug.This fluid passes through these channels, into the core, dissolving thedrug, and the resultant solution of drug may diffuse out in a controlledmanner. This may permit both controlled dissolution and absorption. Anadvantage of this system is that the drug releasing pores of the tabletare distributed over substantially the entire surface of the tablet.This facilitates uniform drug absorption and reduces aggressiveunidirectional drug delivery. MODAS represents a very flexible dosageform in that both the inner core and the outer semipermeable membranemay be altered to suit the individual delivery requirements of a drug.In particular, the addition of excipients to the inner core may help toproduce a micro-environment within the tablet that facilitates morepredictable release and absorption rates. The addition of an immediaterelease outer coating may allow for development of combination products.

Additionally, PRODAS may be used to deliver the ADA inhibitor accordingto the invention. PRODAS is a multi particulate drug delivery technologybased on the production of controlled release mini tablets in the sizerange of 1.5 to 4 mm in diameter. The PRODAS technology is a hybrid ofmulti particulate and hydrophilic matrix tablet approaches, and mayincorporate, in one dosage form, the benefits of both these drugdelivery systems.

In its most basic form, PRODAS involves the direct compression of animmediate release granulate to produce individual mini tablets thatcontain adenosine analogs according to the invention. These mini tabletsare subsequently incorporated into hard gels and capsules that representthe final dosage form. A more beneficial use of this technology is inthe production of controlled release formulations. In this case, theincorporation of various polymer combinations within the granulate maydelay the release rate of drugs from each of the individual minitablets. These mini tablets may subsequently be coated with controlledrelease polymer solutions to provide additional delayed releaseproperties. The additional coating may be necessary in the case ofhighly water soluble drugs or drugs that are perhaps gastro-irritantswhere release can be delayed until the formulation reaches more distalregions of the gastrointestinal tract. One value of PRODAS technologylies in the inherent flexibility to formulation whereby combinations ofmini tablets, each with different release rates, are incorporated intoone dosage form. As well as potentially permitting controlled absorptionover a specific period, this also may permit targeted delivery of drugto specific sites of absorption throughout the gastrointestinal tract.Combination products also may be possible using mini tablets formulatedwith different active ingredients.

DUREDAS is a bilayer tableting technology that may be used in thepractice of the invention. DUREDAS was developed to provide for twodifferent release rates, or dual release of a drug from one dosage form.The term bilayer refers to two separate direct compression events thattake place during the tableting process. In a preferable embodiment, animmediate release granulate is first compressed, being followed by theaddition of a controlled release element which is then compressed ontothis initial tablet. This may give rise to the characteristic bilayerseen in the final dosage form.

The controlled release properties may be provided by a combination ofhydrophilic polymers. In certain cases, a rapid release of the ADAinhibitor according to the invention may be desirable in order tofacilitate a fast onset of therapeutic affect. Hence one layer of thetablet may be formulated as an immediate release granulate. By contrast,the second layer of the tablet may release the drug in a controlledmanner, preferably through the use of hydrophilic polymers. Thiscontrolled release may result from a combination of diffusion anderosion through the hydrophilic polymer matrix.

A further extension of DUREDAS technology, is the production ofcontrolled release combination dosage forms. In this instance, twodifferent ADA inhibitors according to the invention may be incorporatedinto the bilayer tablet and the release of drug from each layercontrolled to maximize therapeutic affect of the combination.

One preferable example of coadministration is the combination ofdeoxyadenosines with pentostatin. This combination may operatesynergistically, to obtain a differential effect over either of thetherapeutic agents administered separately. It has been reported thatpentostatin enhances the clinical anti-HIV activity of related adenosineanalogs presumably due to prevention of degradation of the adenosineanalogs by adenosine deaminase. G. S. Ahluwalia, et al., “Enhancement by2′-deoxycoformycin of the 5″-Phosphorylation and Anti-Humanimmunodeficiency virus activity of 2′3′-dideoxyadenosine and2′-beta-fluor-2′,3′-dideoxyadenosine”, Molec. Pharmacol. 46:1002-1008(1994).

Furthermore, the ADA inhibitor may be administered or coadministeredwith conventional pharmaceutical excipients and additives. Theseinclude, but are not limited to, gelatin, natural sugars such as rawsugar or lactose, lecithin, pectin, starches (for example corn starch oramylose), dextran, polyvinyl pyrrolidone, polyvinyl acetate, gum arabic,alginic acid, tylose, talcum, lycopodium, silica gel (for examplecolloidal), cellulose, cellulose derivatives (for example celluloseethers in which the cellulose hydroxy groups are partially etherifiedwith lower saturated aliphatic alcohols and/or lower saturated,aliphatic oxyalcohols, for example methyl oxypropyl cellulose, methylcellulose, hydroxypropyl methyl cellulose, hydroxypropyl methylcellulose phthalate), fatty acids as well as magnesium, calcium oraluminum salts of fatty acids with 12 to 22 carbon atoms, in particularsaturated (for example stearates), emulsifiers, oils and fats, inparticular vegetable (for example, peanut oil, castor oil, olive oil,sesame oil, cottonseed oil, corn oil, wheat germ oil, sunflower seedoil, cod liver oil, in each case also optionally hydrated); glycerolesters and polyglycerol esters of saturated fatty acids C₁₂H₂₄O₂ toC₁₈H₃₆O₂ and their mixtures, it being possible for the glycerol hydroxygroups to be totally or also only partly esterified (for example mono-,di- and triglycerides); pharmaceutically acceptable mono- or multivalentalcohols and polyglycols such as polyethylene glycol and derivativesthereof, esters of aliphatic saturated or unsaturated fatty acids (2 to22 carbon atoms, in particular 10-18 carbon atoms) with monovalentaliphatic alcohols (1 to 20 carbon atoms) or multivalent alcohols suchas glycols, glycerol, diethylene glycol, pentacrythritol, sorbitol,mannitol and the like, which may optionally also be etherified, estersof citric acid with primary alcohols, acetic acid, urea, benzylbenzoate, dioxolanes, glyceroformals, tetrahydrofurfuryl alcohol,polyglycol ethers with C₁-C₁₂-alcohols, dimethylacetamide, lactamides,lactates, ethylcarbonates, silicones (in particular medium-viscouspolydimethyl siloxanes), calcium carbonate, sodium carbonate, calciumphosphate, sodium phosphate, magnesium carbonate and the like.

Other auxiliary substances that may be used are those which causedisintegration (so-called disintegrants), such as: cross-linkedpolyvinyl pyrrolidone, sodium carboxymethyl starch, sodium carboxymethylcellulose or microcrystalline cellulose. Conventional coating-substancesmay also be used to produce the oral dosage form. Those that may forexample be considered are: polymerizates as well as copolymerizates ofacrylic acid and/or methacrylic acid and/or their esters;copolymerizates of acrylic and methacrylic acid esters with a lowerammonium group content (for example EudragitR RS), copolymerizates ofacrylic and methacrylic acid esters and trimethyl ammonium methacrylate(for example EudragitR RL); polyvinyl acetate; fats, oils, waxes, fattyalcohols; hydroxypropyl methyl cellulose phthalate or acetate succinate;cellulose acetate phthalate, starch acetate phthalate as well aspolyvinyl acetate phthalate, carboxy methyl cellulose; methyl cellulosephthalate, methyl cellulose succinate, -phthalate succinate as well asmethyl cellulose phthalic acid half ester; zein; ethyl cellulose as wellas ethyl cellulose succinate; shellac, gluten; ethylcarboxyethylcellulose; ethacrylate-maleic acid anhydride copolymer; maleic acidanhydride-vinyl methyl ether copolymer; styrol-maleic acidcopolymerizate; 2-ethyl-hexyl-acrylate maleic acid anhydride; crotonicacid-vinyl acetate copolymer; glutaminic acid/glutamic acid estercopolymer; carboxymethylethylcellulose glycerol monooctanoate; celluloseacetate succinate; polyarginine.

Plasticizing agents that may be considered as coating substances are:citric and tartaric acid esters (acetyl-triethyl citrate, acetyltributyl-, tributyl-, triethyl-citrate); glycerol and glycerol esters(glycerol diacetate, -triacetate, acetylated monoglycerides, castoroil); phthalic acid esters (dibutyl-, diamyl-, diethyl-, dimethyl-,dipropyl-phthalate), di-(2-methoxy- or 2-ethoxyethyl)-phthalate,ethylphthalyl glycolate, butylphthalylethyl glycolate andbutylglycolate; alcohols (propylene glycol, polyethylene glycol ofvarious chain lengths), adipates (diethyladipate, di-(2-methoxy- or2-ethoxyethyl)-adipate; benzophenone; diethyl- and diburylsebacate,dibutylsuccinate, dibutyltartrate; diethylene glycol dipropionate;ethyleneglycol diacetate, -dibutyrate, -dipropionate; tributylphosphate, tributyrin; polyethylene glycol sorbitan monooleate(polysorbates such as Polysorbar 50); sorbitan monooleate.

As mentioned above, the ADA inhibitor may be orally administered orcoadministered in a liquid dosage form. For the preparation of solutionsor suspensions it is, for example, possible to use water, particularlysterile water, or physiologically acceptable organic solvents, such asalcohols (ethanol, propanol, isopropanol, 1,2-propylene glycol,polyglycols and their derivatives, fatty alcohols, partial esters ofglycerol), oils (for example peanut oil, olive oil, sesame oil, almondoil, sunflower oil, soya bean oil, castor oil, bovine hoof oil),paraffins, dimethyl sulphoxide, triglycerides and the like.

In the case of drinkable solutions the following substances may be usedas stabilizers or solubilizers: lower aliphatic mono- and multivalentalcohols with 2-4 carbon atoms, such as ethanol, n-propanol, glycerol,polyethylene glycols with molecular weights between 200-600 (for example1 to 40% aqueous solution), diethylene glycol monoethyl ether,1,2-propylene glycol, organic amides, for example amides of-aliphaticC₁-C₆-carboxylic acids with ammonia or primary, secondary or tertiaryC₁-C₄-amines or C₁-C₄-hydroxy amines such as urea, urethane, acetamide,N-methyl acetamide, N,N-diethyl acetamide, N,N-dimethyl acetamide, loweraliphatic amines and diamines with 2-6 carbon atoms, such as ethylenediamine, hydroxyethyl theophylline, tromethamine (for example as 0.1 to20% aqueous solution), aliphatic amino acids.

In preparing the inventive compositions, it is possible to use known andconventional solubilizers or emulsifiers. Solubilizers and emulsifiersthat may for example be used are: polyvinyl pyrrolidone, sorbitan fattyacid esters such as sorbitan trioleate, phosphatides such as lecithin,-acacia, tragacanth, polyoxyethylated sorbitan monooleate and otherethoxylated fatty acid esters of sorbitan, polyoxyethylated fats,polyoxyethylated oleotriglycerides, linolizated oleotriglycerides,polyethylene oxide condensation products of fatty alcohols, alkylphenolsor fatty acids or also 1-methyl-3-(2-hydroxyethyl)imidazolidone-(2). Inthis context, polyoxyethylated means that the substances in questioncontain polyoxyethylene chains, the degree of polymerization of whichgenerally lies between 2 and 40 and in particular between 10 and 20.

Polyoxyethylated substances of this kind may for example be obtained byreaction of hydroxyl group-containing compounds (for example mono- ordiglycerides or unsaturated compounds such as those containing oleicacid radicals) with ethylene oxide (for example 40 Mol ethylene oxideper 1 Mol glyceride).

Examples of oleotriglycerides are olive oil, peanut oil, castor oil,sesame oil, cottonseed oil, corn oil. See also Dr. H. P. Fiedler“Lexikon der Hillsstoffe für Pharmazie, Kostnetik und angrenzendeGebiete” 1971, pages 191-195.

It is also possible to add preservatives, stabilizers, buffersubstances, flavor correcting agents, sweeteners, colorants,antioxidants and complex formers and the like. Complex formers which maybe for example be considered are: chelate formers such as ethylenediamine retrascetic acid, nitrilotriacetic acid, diethylene triaminepentacetic acid and their salts.

It may optionally be necessary to stabilize the ADA inhibitor withphysiologically acceptable bases or buffers to a pH range ofapproximately 6 to 9. Preference may be given to as neutral or weaklybasic a pH value as possible (up to pH 8).

In some dosage forms, it may be useful to include antioxidants orpreservatives. Antioxidants that may for example be used are sodiumsulphite, sodium hydrogen sulphite, sodium metabisulphite, ascorbicacid, ascorbylpalmitate, -myristate, -stearate, gallic acid, gallic acidalkyl ester, butylhydroxyamisol, nordihydroguaiaretic acid, tocopherolsas well as synergists (substances which bind heavy metals throughcomplex formation, for example lecithin, ascorbic acid, phosphoric acidethylene diamine tetracetic acid, citrates, tartrates). Addition ofsynergists substantially increases the antioxygenic effect of theantioxidants.

Preservatives that may for example be considered are sorbic acid,p-hydroxybenzoic acid esters (for example lower alkyl esters), benzoicacid, sodium benzoate, trichloroisobutyl alcohol, phenol, cresol,benzethonium chloride, chlorhexidine and formalin derivatives.

The various oral dosage forms can be prepared according to conventionalprocedures. For example, tablets can be prepared according to commontableting procedures. Capsules can be prepared according to conventionalencapsulating procedures. Liquid dosage forms may be supplied as amade-up vial, or may be supplied in a lyophilized state for dilutionjust prior to administration. Controlled release dosage forms may beprepared according to the particular dosage form being used, as isdiscussed briefly above.

3) Parenteral Formulation

Alternatively, the ADA inhibitor such as pentostatin may be formulatedfor parenteral administration such as intravenous infusion. In apreferred embodiment, pentostatin is parenterally administered to thepatient at a lower dose than the standard dose used for treating haircell leukemia (4 mg/m²). Thus, pentostatin may be supplied as a sterile,apyrogenic, lyopholized powder in single dose vials at about 1-5 mg/vialand more preferably at 2-4 mg/vial. The vials may also containexcipients such as mannitol. Pentostatin contained in the single dosevial can be reconstituted by using sterile water, saline or otherinfusion fluid prior to infusion. The pH of the final infusion fluidcontaining low dose pentostatin is preferably maintained between 7.0 and8.5 by addition of sodium hydroxide or hydrochloric acid.

3. Method of Use and Dosing Regimen

The ADA inhibitor may be used to treat patients with various forms ofGVHD including acute and chronic GVHD that is either naive or refractoryto conventional immunosuppressive agents such as steroids andcyclosporin A. The ADA inhibitor may also be used as prophylaxis toprevent onset of GVHD by pretreating the transplant recipient prior tothe transplantation and/or treating the recipient within a certain timewindow post transplantation.

1) Treatment of GVHD

In one embodiment, a method is provided for treating a patient sufferingfrom GVHD. The method comprises administering to the GVHD patient acomposition including an ADA inhibitor.

Dosage amounts and frequency will vary according to the particular ADAinhibitor, the dosage form, and individual patient characteristics.Generally speaking, determining the dosage amount and frequency for aparticular ADA inhibitor (e.g., pentostatin), dosage form, andindividual patient characteristic can be accomplished using conventionaldosing studies, coupled with appropriate diagnostics. In preferableembodiments, the dosage frequency ranges from daily to monthly doses,more preferably biweekly doses. In other preferable embodiments, thedosage amount ranges from about 0.02 mg/m² to about 20 mg/m², morepreferably about 0.05 mg/m² to about 5 mg/m², and most preferably 0.1mg/m² to about 1 mg/m².

A dosage amount below 1 mg/m² is considered to be much lower than thestandard dose (4 mg/m² once every 2 weeks) used for the treatment ofhairy cell leukemia. According to the present invention, lowering thedosage amount of pentostatin should minimize myelosuppression associatedwith high dose of this antimetabolic drug and yet can still specificallysuppress the GVHD-causing T-lymphocyte mediated immune response.

In a particular embodiment, an ADA inhibitor such as pentostatin is usedto treat patients that have acute Graft vs Host Disease (aGVHD) butfailed at least one immunosuppressive regimen such as a regimenincluding steroids such as prednisone and methylprednisolone,cyclophosphamide, cyclosporin A, FK506, thalidomide, azathioprine, anddaclizumab. For example, hematopoietic stem cell transplant (HSCT)patients manifesting grade 2 or greater aGVHD, who have failed torespond to treatment with at least 2 mg/kg of methylprednisolone orequivalent corticosteroid or other salvage therapy, can be treated withpentostatin.

In a preferred embodiment, the HSCT patient with steroid-refractoryaGVHD is treated with pentostatin according to the following plan. Thepatient is treated with 0.25-1 mg/m²/day as a 20 minute intravenous (IV)infusion on days 1, 2 and 3. The patient's response to the treatment ismonitored by following for improvement in the skin, mouth, fascia, andliver. The same dose may be repeated after 14 days if there w asresponse to the first 3 days of treatment that was incomplete or thatrecurred after a full or partial response.

Pentostatin may also be administered orally to achieve similar plasmalevels as those via IV route of administration. However, for aGVHDpatients with severely damaged gastrointestinal (GI) tract IV infusionmay be the preferred route of administration.

In another embodiment, an ADA inhibitor such as pentostatin is used totreat steroid-refractory chronic Graft vs Host Disease (cGVHD). Forexample, recipients of allogeneic HSCT developing cGVHD who have failedto respond to treatment with at least 2 mg/Kg of methylprednisolone orequivalent corticosteroid or other salvage therapy, can be treated withpentostatin via IV or oral administration.

In a variation of this embodiment, pentostatin is orally administratedto the cGVHD patient at a dose between about 1-10 mg/m², preferablybetween about 2-6 mg/m², and more preferably between about 2-4 mg/m²each day for 3 consecutive days each month. For an average adult, a doseof 4 mg/m² would be approximately 6-8 mg/day (one and a half or two 4 mgtablets or capsules).

2) GVHD Prophylaxis

An ADA inhibitor such as pentostatin can also be used as a prophylaxisto prevent onset of GVHD or to reduce the effects of GVHD.

Pentostatin may be administered as a GVHD prophylaxis parenterally ororally to a transplant recipient within a predetermined time windowbefore or after the transplantation.

In one embodiment, pentostatin may be administered orally to therecipient on days −3 or −2 (i.e., 3 or 2 days before thetransplantation) as part of a non-myeloablative conditioning regimen,then followed by transplantation such as hematopoietic stem cellinfusion. Alternatively, pentostatin may be administered to therecipient by IV infusion at a dose lower than 2 mg/m², preferably at adose lower than 1 mg/m².

In myeloablative conditioning regimen, the transplant recipient may betreated with pentostatin p.o. (i.e., via oral administration) at 0.5-2.0mg/m² on days −14, −13, −12 and −3, −2, −1 with high dosecyclophosphamide and/or busulfan and/or melphalan and/or 1200-1800 cGyirradiation prior to stem cell infusion. Post transplantation therecipient may be continuously treated with pentostatin, on days +8 and+15 preferably iv at a lower dose such as 1-2 mg/m² since patientsreceiving myeloablative conditioning might not be able to tolerate anoral dosage form on days +8 and +15 due to severe mucositis.

In another embodiment, pentostatin may be administered as a GVHDprophylaxis to a transplant recipient after the transplantation. Forexample, for standard (i.e. myeloablative) transplant ornon-myeloablative stem cell transplant (NST) where pentostatin is notused in the conditioning regimen, pentostatin is administered to thetransplant recipient at 0.5-1.5 mg/m²/day on days +8, +15, +22 and +30following stem cell infusion.

4. Combination Therapy for GVHD

Besides use in a single-agent treatment or prevention of GVHD, the ADAinhibitor such as pentostatin can also be used in a combination therapyfor acute or chronic GVHD. The combination therapy may have synergistictherapeutic effects on the patients and thus requires lower amount ofpentostatin and the other agent used in conjunction to achievesatisfactory therapeutic efficacy. As a result, potential side effectsassociated with high dose of drugs, such as myelosuppression, arereduced and the patient's quality of life is improved.

Various other therapeutic agents may be combined with pentostatin forthe treatment or prevention of GVHD. The other therapeutic agentsinclude, but are not limited to, immunosuppressive agents such assteroids (e.g., prednisone and methylprednisolone), cyclophosphamide,cyclosporin A, FK506, thalidomide, azathioprine, monoclonal antibodies(e.g., Daclizumab (anti-interleukin (IL)-2), Infliximab (anti-tumornecrosis factor), MEDI-205 (anti-CD2), abx-cbl (anti-CD147)), andpolyclonal antibodies (e.g., ATG (anti-thymocyte globulin)).

For example, pentostatin may be combined with a steroid such asmethylprednisolone to treat aGVHD. However, such a combination may betoo broadly immunosuppressive to render the patient more susceptible toopportunistic infection.

For the treatment of acute GVHD, pentostatin may preferably be combinedwith monoclonal antibodies which specifically target T-cells such asInfliximab, Daclizumab, M-EDI-205, or abx-cbl. The monoclonal antibodymay be administered at the FDA-approved dosage and by its standard routeof administration (e.g., IV), followed by oral or parenteraladministration of pentostatin as described in detail in Section 3 above.

For the treatment of chronic GVHD, pentostatin may be combined withthalidomide. Pentostatin may also be used in conjunction with otherimmunosuppressive agents as prophylaxis for GVHD post transplantation.For example, the recipient of bone marrow transplant may be treated withpentostatin in conjunction with a standard post infusion regimenincluding mini-methotrexate at 5 mg/m² (as opposed to the conventionaldose at 10-15 mg/m²), cyclosporine A (5-6 mg/Kg/d IV or 10-18 mg/Kg/dorally) and FK506 (0.05-0.1 mg/Kg/d IV or 0.15-0.3 mg/Kg/d orally).

In addition, pentostatin may be used in conjunction with other types oftherapy as prophylaxis for GVHD prior to transplantation. For example,the recipient of bone marrow transplant may be pretreated withpentostatin in conjunction with TBI (radiation), phototherapy,melphalan, cyclophosphamide or ATG to prevent the onset of GVHD.

5. Ex Vivo Treatment of Transplants Using ADA Inhibitors

In yet another aspect, the invention relates to a method of ex vivo orin vitro treatment of blood derived cells, bone marrow transplants, orother organ transplants. The method comprises treating the blood derivedcells, bone marrow transplants, or other organ transplants with an ADAinhibitor (e.g., pentostatin) in an effective amount such thatactivities of T-lymphocytes therein are substantially inhibited,preferably by at least 50% reduction in activity, more preferably by atleast 80% reduction in activity, and most preferably by at least 90%reduction in activity.

The invention is practiced in an in vitro or ex vivo environment. All ofthe discussion above regarding clinical treatment or prevention of GVHDthat is relevant to an in vitro or ex vivo environment applies to thispractice. In a particular embodiment, practice of an in vitro or ex vivoembodiment of the invention might be useful in the practice of immunesystem transplants, such as bone marrow transplants or peripheral stemcell procurement. In such procedures, the ADA inhibitor might be used,as generally described above, to treat the transplant material toinactivate T-lymphocytes therein so that the T-lymphocyte mediatedimmune response is suppressed upon transplantation.

For example, the ADA inhibitor may be added to a preservation solutionfor an organ transplant in an amount sufficient to inhibit activity ofT-lymphocytes of the organ. Such a preservation solution maybe suitablefor preservation of different kind of organs such as heart, kidney andliver as well as tissue therefrom. An example of commercially availablepreservation solutions is Plegisol (Abbott), and other preservationsolutions named in respect of its origins include the UW-solution(University of Wisconsin), the Stanford solution and the ModifiedCollins solution (J. Heart Transplant (1988) Vol. 7(6):456-4467). Thepreservation solution may also contain conventional co-solvents,excipients, stabilizing agents and/or buffering agents.

The dosage form of the ADA inhibitor may be a liquid solution ready foruse or intended for dilution with a preservation solution.Alternatively, the dosage form may be lyophilized or power filled priorto reconstitution with a preservation solution. The lyophilizedsubstance may contain, if suitable, conventional excipients.

The preservation solution or buffer containing an ADA inhibitor (e.g.,pentostatin) may also be used to wash or rinse an organ transplant priorto transplantation or storage. For example, a preservation solutioncontaining pentostatin may be used to flush perfuse an isolated heartwhich is then stored at 4° C. in the preservation solution.

In another embodiment, practice of the invention might be used tocondition organ transplants prior to transplantation. Prior totransplantation pentostatin may be added to the washing buffer to ridthe transplant of active T-lymphocytes. In this way, the risk ofdeveloping acute GVHD upon transplantation should be significantlyreduced, and the host is not only protected from GVHD but also frompotential side effects of pentostatin.

The concentration of the ADA inhibitor in the preservation solution orwash buffer may vary according to the type of transplant. For example,pentostatin at 1 μM may be used to treat an isolated heart prior totransplantation.

Other applications in vitro or ex vivo using an ADA inhibitor will occurto one of skill in the art and are therefore contemplated as beingwithin the scope of the invention.

1-38. (canceled)
 39. A method for ex vivo treatment of a tissue or organtransplant having T-lymphocyte activity comprising contacting the tissueor organ transplant with a composition comprising an effective,T-lymphocytes-inhibiting amount of an adenosine deaminase.
 40. Themethod of claim 39, wherein the tissue or organ transplant is selectedfrom the group consisting of stem cells, bone marrow, heart, liver,kidney, lung pancreas, small intestine, cornea and skin.
 41. The methodof claim 39, wherein the adenosine deaminase inhibitor is selected fromthe group consisting of pentostatin, fludarabine monophosphate andcladribine.
 42. The method of claim 39, further comprising inhibitingthe activity of T-lymphocytes in the tissue or organ transplant by atleast about 50%.
 43. The method of claim 39, further comprisinginhibiting the activity of T-lymphocytes in the tissue or organtransplant by at least about 80%.
 44. The method of claim 39, furthercomprising inhibiting the activity of T-lymphocytes in the tissue ororgan transplant by at least about 90%.
 45. The method of claim 39,wherein contacting the tissue or organ transplant includes storing thetissue or organ transplant in a preservation solution containingpentostatin.
 46. The method of claim 45, wherein the preservationsolution is one of or a mixture of Plegisol, University of Wisconsinsolution, Stanford solution, and Modified Collins Solution.
 47. Themethod of claim 39, further comprising washing the tissue or organtransplant with a buffer containing pentostatin prior totransplantation.
 48. The method of claim 47, wherein the tissue or organtransplant is an isolated heart transplant.
 49. The method of claim 48,wherein washing the isolated heart transplant includes flush perfusingthe heart with the buffer, and wherein the buffer comprises about 0.1-10μM pentostatin.
 50. A method for reducing T-lymphocyte activity in atissue or organ transplant having such activity comprising: contacting,ex vivo, the tissue or organ transplant with a composition comprising aneffective, T-lymphocytes-activity-reducing amount of an adenosinedeaminase selected from the group consisting of pentostatin, fludarabinemonophosphate and cladribine.
 51. The method of claim 50, wherein thetissue or organ transplant is selected from the group consisting of stemcells, bone marrow, heart, liver, kidney, lung pancreas, smallintestine, cornea and skin.
 52. The method of claim 50, furthercomprising reducing the activity of T-lymphocytes in the tissue or organtransplant by at least about 50%.
 53. The method of claim 50, furthercomprising reducing the activity of T-lymphocytes in the tissue or organtransplant by at least about 80%.
 54. The method of claim 50, furthercomprising reducing the activity of T-lymphocytes in the tissue or organtransplant by at least about 90%.
 55. The method of claim 50, whereincontacting the tissue or organ transplant includes storing the tissue ororgan transplant in a preservation solution containing pentostatin. 56.The method of claim 55, wherein the preservation solution is selectedfrom the group consisting of Plegisol, University of Wisconsin solution,Stanford solution, and Modified Collins Solution.
 57. The method ofclaim 50, further comprising washing the tissue or organ transplant witha buffer containing pentostatin prior to transplantation.
 58. The methodof claim 57, wherein the tissue or organ transplant is an isolated hearttransplant.
 59. The method of claim 58, wherein washing the isolatedheart transplant includes flush perfusing the heart with the buffer, andwherein the buffer comprises about 0.1-10 μM pentostatin.
 60. A methodfor preventing increased T-lymphocyte activity in a tissue or organtransplant having such activity comprising: contacting, ex vivo, thetissue or organ transplant with a composition comprising an effectiveamount of an adenosine deaminase selected from the group consisting ofpentostatin, fludarabine monophosphate and cladribine.
 61. The method ofclaim 60, wherein the tissue or organ transplant is selected from thegroup consisting of stem cells, bone marrow, heart, liver, kidney, lungpancreas, small intestine, cornea and skin.